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The present invention relates to an automated system for simultaneously performing a plurality of assays of test samples, detecting the results of the assays, and collecting and storing the data. The system comprises three major components: a detection system, a robotic fluid handling system, and a computer controlled data acquisition and data analysis system. The present invention further relates to a method for simultaneously performing a plurality of fluorescence assays, detecting the plurality of assays, and collecting, storing and analyzing the data.
Signal-generating techniques are often employed to detect chemical reactions, biological events, and physical and chemical properties of a sample. Typically, the signal is in the form of radiation (e.g., light, color, fluorescence, luminescence, particle emissions) and either is a component/product of the reaction or is generated upon interaction of a component/product with an added indicator moiety.
An example of the use of signal generation to detect a biological substance is the quantitation of an antigen in biological samples using enzyme-linked immunosorbent assays (ELISA). In these assays, a sample is exposed to an enzyme-conjugated antibody capable of binding to the specific antigen to be detected. The conjugated enzyme is one that catalyzes a reaction which generates a signal (e.g., color, fluorescence, luminescence) that can be directly correlated with the amount of antigen in the sample. This type of assay, in which the property to be measured is constant and the signal is sustained, is referred to as an endpoint assay. Thus, in these assays, the signal is allowed to develop over time, and then a single signal measurement is taken after the reaction is complete in order to quantify the property.
In contrast to attributes that can be measured in endpoint assays, there are many properties, reactions and biological events that are dynamic and transient and/or rapidly occurring. For example, many cellular processes are rapid and transient in nature. Cells receive stimuli from the environment and must respond immediately for proper function and survival. Modulation of cell receptors and ion channels by binding of ligands can result in cellular responses such as changes in the levels of intracellular second messengers (Ca2+, cyclic nucleotides, etc.). For instance, activation of a cell surface calcium channel upon binding of a ligand causes the channel to open and results in a rapid inward flux of calcium that transiently increases the intracellular Ca2+ concentration which rapidly declines to preactivation concentrations. If the cell has been pre-loaded with a Ca2+-sensitive fluorescent indicator, the change in intracellular Ca2+ appears as a rapid increase and then decrease in fluorescence of the cell.
Because signal-generation techniques can provide information regarding the actual functioning of a cell, it is desirable to attempt to apply these methods to the identification of compounds that influence cellular activities (e.g., potential drugs that affect cell function through interaction with cell receptors, ion channels or enzymes). However, in drug screening procedures, large number of compounds are tested for cell modulation before even a small number are identified as potential drugs. The problems faced in using signal-generation techniques to detect and measure such transient and/or rapidly occurring phenomena in a single assay are only compounded in attempting to apply these techniques to the simultaneous performance of multiple assays for rapid screening of thousands of compounds.
For instance, the signal generated in these assays is rapidly occurring and transient, as is the phenomenon itself. Thus, in these assays, if initiation of the reaction or event (e.g., activation of the calcium channels by addition of ligand) is not coordinated with almost immediate signal detection in a dynamic fashion, the signal may reach a maximum and diminish before it is detected. In order to perform large-scale compound screening, coordination of sample handling and signal detection must be accomplished for many assays simultaneously. Furthermore, it is desirable to obtain a real-time record of each event until it has progressed to a point beyond that of maximum signal change. Thus, the duration, as well as the timing, of signal measurement poses an additional complication in these assays since the signal must be measured essentially constantly.
Accuracy of signal measurement is particularly critical in high-throughput screening assays of thousands of compounds. The need to perform a multitude of individual compound tests in a limited amount of time prohibits replicate assays of each compound. Additionally, sensitivity of signal detection presents another difficulty in signal-based assays of these phenomena. The signal changes accompanying these reactions or events are not only transient changes in the relative levels of the signal (i.e., increases in signal above a baseline level of signal), but may also be of relatively small magnitude. In large-scale drug screening, these transient, relatively small signal changes must be detected in multiple assays simultaneously.
Consequently, there is little margin for error in each single compound assay; an erroneous signal measurement by the detection system could result in elimination of a viable drug from further consideration. Additionally, signal measurement accuracy and sensitivity is essential in detecting small but significant differences in cellular responses to varying doses of compounds and in the response generated by an unknown compound as compared to a standard known drug.
Thus, there is a need for signal detection instrumentation that enables fully automated, high-volume assays of rapid, transient phenomena with sufficient sensitivity and the degree of accuracy required for applications such as drug screening.
The present invention provides an integrated sample handling and detection system that enables simultaneous preparation and performance of multiple assays of rapidly occurring, transient phenomena in a plurality of individual wells of a test plate; imaging of the assays with sufficient sensitivity and a high degree of accuracy continuously in real time over a period of time; and collection, storage, and analysis of the imaging data. The detection system and method of the present invention enable automated assays of large numbers of test samples quickly, efficiently, accurately, and economically.
The system according to the present invention is capable of accurately and simultaneously imaging a large number of potentially low-intensity, rapid, transient reactions. The system includes a robotic fluid handling system for automated delivery of liquids to wells of a test plate; a detection system for detecting the assays as they are performed; and a computer-controlled data acquisition and analysis system for controlling the operation of the entire system and for collecting and analyzing imaging data.
A preferred embodiment of the system of the present invention includes an apparatus for simultaneously performing a plurality of fluorescence assays including a plate containing a plurality of wells; a distributor for simultaneously distributing a predetermined amount of a liquid to each of the plurality of wells; an excitation source for simultaneously exposing the wells to excitation radiation; a detector for simultaneously detecting fluorescence emitted from each of the plurality of wells continuously in real time over a predetermined period of time; and a computerized controller for simultaneously controlling and coordinating the distributor, the excitation source, and the detector.
A second preferred embodiment of the system of the present invention includes an apparatus for simultaneously performing a plurality of signal-based assays including a plate containing a plurality of wells; a distributor for simultaneously distributing a predetermined amount of a liquid to a number of the plurality of wells; a detector for simultaneously detecting emissions emitted from each of the plurality of wells over a predetermined period of time; and a computerized controller for automatically and simultaneously coordinating the distributor and the detector.
A particularly preferred aspect of the system provides for increased accuracy of signal measurement by taking the ratio of the signals measured after excitation with light of a first wavelength and the signals measured after excitation with light of a second different wavelength. The ratio of two emitted light measurements can be a more accurate determination of the actual emitted light than single absolute measurements because the ratio cancels the effects of instrument drift, transient changes in instrument sensitivity and changes in cell volume or fluorescent indicator concentration, each of which may be mistaken for a change in the attribute being measured.
A third embodiment of the system of the present invention includes a plate containing a plurality of wells; a distributor for simultaneously distributing a predetermined amount of a liquid to each of the plurality of wells; a detector for simultaneously detecting optical emissions emitted from each of the plurality of wells over a predetermined period of time, wherein the detector includes a single imager for optically imaging the plurality of wells, the detector creating a time series of pixel images of each of the plurality of wells to determine an amount of optical emissions emitted from each of the plurality of wells over the predetermined period of time; a computer processor for acquiring, processing, and storing optical emissions data detected by the detector; and a computerized controller for simultaneously controlling and coordinating the distributor, the detector, and the computer processor.
A method according to the present invention comprises the steps of simultaneously distributing a predetermined amount of a liquid to a number of a plurality of wells; simultaneously exposing the wells to excitation radiation; simultaneously detecting fluorescence emitted from the plurality of wells over a predetermined period of time using a detector; processing fluorescence data detected by the detector; and simultaneously controlling and coordinating the distribution, excitation, and detection using a computerized controller.
Another method for performing simultaneous assays according to the present invention includes the steps of simultaneously distributing a predetermined amount of a liquid to a number of the plurality of wells; simultaneously detecting emissions emitted from the plurality of wells over a predetermined period of time using a detector; processing emissions data detected by the detector; and simultaneously controlling and coordinating the distribution and the detection using a computerized controller.
The method according to the present invention may be used, for example, for drug screening, wherein compound samples are assayed to identify compositions having the ability to activate, potentiate, or inhibit ion channels and/or receptors of a cell that, when activated, directly or indirectly contribute to a detectable change in the level of a predetermined ion in the cell. When used for drug screening, the method of the present invention includes the steps of providing each of a plurality of wells with viable cells having functional ion channels and/or receptors which, when activated, are capable of directly or indirectly causing a detectable change in a concentration of a predetermined ion in the viable cells, wherein the viable cells contain an amount of an ion-sensitive indicator sufficient to detect a change, if any, in the concentration of the predetermined ion; simultaneously distributing a predetermined amount of a putative ion channel-activating or receptor-activating, -potentiating or -inhibiting compound being tested for its ability to activate, potentiate or inhibit the ion channel or receptor to each of the plurality of wells; simultaneously detecting optical emissions emitted by the ion-sensitive indicator in each of the plurality of wells over a predetermined period of time using a detector consisting of a single imager for optically imaging the plurality of wells, the detector creating a time series of pixel images of each of the plurality of wells to determine an amount of optical emissions of the ion-sensitive indicator in the plurality of wells over the predetermined period of time; processing optical emissions data detected by the detector; and simultaneously controlling and coordinating the distribution, excitation, detection, and processing using a computerized controller.
When the method according to the present invention is used for screening compounds to identify compositions having the ability to inhibit or block ion channels and/or receptors of a cell (e.g., antagonist compounds), the test compound is added to the wells before or simultaneously with a known activator of the ion channels and/or receptors. The signal detected from the wells is compared to that detected from identical wells to which only the known activator is added in the absence of the test compound or from wells containing cells identical to the ion channel and/or receptor-containing cells except that they do not express the ion channels and/or receptors.
The method according to the present invention may also be used for screening cell lines to identify those that express functional ion channels and/or receptors. In these assays, known modulators of the ion channels and/or receptors are added to the wells containing the test cells, and the signal emitted by the ion-sensitive indicator is measured to determine if the intracellular ion concentration has changed in response to the addition of a known modulator to the cells.